Bisphosphonate (BPs) are potent anti-resorptive agents that are widely used to treat osteoporosis and metastatic bone diseases. Long-term users of BPs are at the higher risk of developing osteonecrosis of the jaw (ONJ). The etiology of bisphosphonate-related osteonecrosis of the jaw (BRONJ) is thought to be due to its inhibitory effects on osteoclasts, bone resorbing cells important for bone remodeling and healing. However, this alone is insufficient to explain the pathophysiology of BRONJ because wound healing in oral environment is a multi-factorial and complex process that requires orchestrated efforts of different cell types including oral mucosal cells. BRONJ commonly occurs at the site of previous tooth extraction or other surgical interventions and is clinically defined as exposed necrotic bone with unhealed and open oral mucosa. Nonetheless, the exact role of oral mucosa in the pathophysiology of BRONJ is not fully understood. To better understand effects of BPs on oral mucosal cells, we established a 3 dimensional (3D) oral mucosal wound healing model and found that BPs drastically inhibit proliferation and migration of keratinocytes. Similar to this ex vivo model, our in vitro studies also showed the marked inhibition of proliferation and migration by BPs specific to primary normal human oral keratinocytes (NHOK). Our recently developed animal model which recapitulates BA-ONJ also revealed the impaired wound closure with underlying exposed and necrotic bone. Based on our preliminary data, we hypothesize that BP directly impairs reepithelialization (e.g., proliferation and migration) of oral mucosa by targeting the mevalonate pathway. To this end, we propose 1) to examine roles of farnesyl pyrophosphate synthase (FPPS) and RhoA in BP-treated NHOK in vitro and ex vivo, and 2) to examine spatial expression patterns of wound healing-associated proteins in vivo using animal model. The clinical outcomes and benefits outweigh the adverse effects of using BPs. Therefore, BPs will continually be used to manage osteoporosis and metastatic bone diseases, and proper treatment and prevention of BRONJ will remain important and relevant clinical issues in dental and medical practices. Our proposal will provide possible explanations as to why BRONJ occurs in oral-cavity specific manner, and will establish a basis for the future clinical applications in managing and treating BRONJ. Successful completions of the current project will likely lead to R01 grant mechanism focusing on the application of findings to the clinical settings. PUBLIC HEALTH RELEVANCE: With the increasing use of bisphosphonates due to its effectiveness in multiple clinical settings, bisphosphonate-related osteonecrosis of the jaw (BRONJ) has been surfaced as a significant dental and medical problem because no definitive prevention and treatment are currently available. Although BRONJ is clinically defined as exposed bone with unhealed and opened overlaying oral mucosa, the exact role of oral mucosa in the pathophysiology of BRONJ is not fully understood. In this proposal, we will investigate direct effects of bisphosphonates on the oral mucosal cells, and our results are expected to contribute significantly on managing long-term bisphosphonate users by improving treatment and prevention modalities.